CHEK2 Contribution to Hereditary Breast Cancer in Non-BRCA Families Alexis Desrichard2, Yannick Bidet2, Nancy Uhrhammer1 and Yves-Jean Bignon1,2*

Desrichard et al. Breast Cancer Research 2011, 13:R119 http://breast-cancer-research.com/content/13/6/R119

RESEARCHARTICLE Open Access CHEK2 contribution to hereditary breast cancer in non-BRCA families Alexis Desrichard2, Yannick Bidet2, Nancy Uhrhammer1 and Yves-Jean Bignon1,2*

Abstract Introduction: Mutations in the BRCA1 and BRCA2 genes are responsible for only a part of hereditary breast cancer (HBC). The origins of “non-BRCA“ HBC in families may be attributed in part to rare mutations in genes conferring moderate risk, such as CHEK2, which encodes for an upstream regulator of BRCA1. Previous studies have demonstrated an association between CHEK2 founder mutations and non-BRCA HBC. However, very few data on the entire coding sequence of this gene are available. Methods: We investigated the contribution of CHEK2 mutations to non-BRCA HBC by direct sequencing of its whole coding sequence in 507 non-BRCA HBC cases and 513 controls. Results: We observed 16 mutations in cases and 4 in controls, including 9 missense variants of uncertain consequence. Using both in silico tools and an in vitro kinase activity test, the majority of the variants were found likely to be deleterious for protein function. One variant present in both cases and controls was proposed to be neutral. Removing this variant from the pool of potentially deleterious variants gave a mutation frequency of 1.48% for cases and 0.29% for controls (P = 0.0040). The odds ratio of breast cancer in the presence of a deleterious CHEK2 mutation was 5.18. Conclusions: Our work indicates that a variety of deleterious CHEK2 alleles make an appreciable contribution to breast cancer susceptibility, and their identification could help in the clinical management of patients carrying a CHEK2 mutation.

Introduction several domains: a SQ/TQ cluster domain, a Forkhead- Breast cancer is one of the main causes of cancer- associated (FHA) domain and a Ser/Thr kinase domain related deaths among women worldwide, with 5% to [11]. In response to DNA double-strand breaks or repli- 10% of cases being due to hereditary risk. However, cative stress, CHEK2 is activated by the kinases ATM mutations in the two major genes, BRCA1 and BRCA2, and ATR [12]. These proteins catalyze the phosphoryla- are found in only 15% to 20% of hereditary breast can- tion of threonine 68 of CHEK2, causing its transient cer (HBC) families [1]. Several studies have reported evi- dimerization via the FHA domain. This leads to CHEK2 dencethatgermlinemutations in other susceptibility trans-autophosphorylation and its full activation [13]. genes, such as ATM, PABL2, BRIP1 and CHEK2,might Activated CHEK2 monomers phosphorylate, in turn, be the predisposing factor in some HBC families [2-5]. numerous downstream substrates, including the P53 In addition, the lower penetrance of these mutations tumor suppressor, CDC25 family proteins and serine suggests that they might act in concert with other her- 988 of BRCA1, activating cell-cycle checkpoints and editary factors [6-10]. increasing DNA repair efficiency [14-17]. These interac- CHEK2 is the human homolog of Rad53 (Saccharo- tions suggest that CHEK2 may also play a role in breast myces cerevisiae)andCds1 (Schizosaccharomyces cancer [14]. pombe). This family of kinases is characterized by Germline CHEK2 mutations are associated with breast cancer in different populations. For example, heterozyg- * Correspondence: [email protected] osity for the well-studied c.1100delC mutation, present 1Laboratoire Diagnostic Génétique et Moléculaire, Centre Jean Perrin, 58 rue in 1.4% of the Finnish population and in 0.2% of the Montalembert, F-63011 Clermont-Ferrand, France Full list of author information is available at the end of the article Polish population, confers a relative risk for developing

© 2011 Desrichard et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Desrichard et al. Breast Cancer Research 2011, 13:R119 Page 2 of 11 http://breast-cancer-research.com/content/13/6/R119

breast tumors of about 2 for women and 10 for men on heparin/lithium using a Genomix blood DNA extrac- [18,19] Likewise, the variant Ile157Thr, present in 5.3% tion kit according to the manufacturer’s instructions of the Finnish population and in 4.8% of the Polish (Talent srl, Trieste, Italy). Samples were resuspended population, confers a relative risk of breast cancer of 1.5 with Tris-ethylenediaminetetraacetic acid (EDTA) (TE) [20,21]. (10mMTris,1mMEDTA,pH8.0).Exons2to10, However, very few groups have studied the entire including intron-exon boundaries, were amplified by CHEK2 gene in HBC [22-25]. It is essential to establish using standard PCR techniques (conditions and primers a causal link between sequence variants and CHEK2 available on request). Because of the multiple copies of function. Little is known about the impact of missense CHEK2 pseudogenes, we used a nested PCR strategy, mutations on protein function, although substitutions in describedpreviouslybySodhaet al. [10], to specifically the FHA domain and the kinase domain have been amplify exons 10 to 14 [30]. Sequence reactions were shown to abolish activity [22,26,27]. In this study, we performed on PCR products purified by ExoSAP-IT screened the whole CHEK2 coding sequence for muta- (Affymetrix, Inc, Santa Clara, CA, USA) using BigDye v3 tions in non-BRCA HBC families and a control popula- reagents (Applied Biosystems/Life Technologies, Foster tion without any family history of breast cancer. Point City, CA, USA) (primers available on request), purified mutations were evaluated by in silico analyses and an in in Sephadex G-50 fine (G5080; Sigma-Aldrich, St Louis, vitro kinase activity test. MO, USA) and analyzed using a 3130xl capillary elec- trophoresis system (Applied Biosystems/Life Technolo- Materials and methods gies). Alignment to the reference sequences was Subjects performed using SeqMan NGen software (DNASTAR, We recruited 507 cases with HBC risk through the Inc, Madison, WI, USA). oncogenetic consultation department at the Centre Jean Perrin (Clermont-Ferrand, France). This group consisted Bioinformatics studies of 258 families with 3 breast cancers in the same famil- For each missense variant, prediction of the impact of ial branch with at least 2 cases related in the first the mutation on the protein was assessed by calculating degree, 237 families with 2 cases of breast cancer in the the SIFT (Sorting Intolerant From Tolerant), Align- same branch with at least 1 breast cancer diagnosed GVGD and PolyPhen-2 (Polymorphism Phenotyping v2) before age 40 years or with bilateral breast cancer, and software tool scores [31-34]. Align-GVGD predictions 12 families with 2 cases of breast cancer and at least 1 and SIFT score were computed using the ortholog align- male breast cancer. One affected patient per HBC family ment of exons 2 to 14 of CHEK2 derived by using Ala- was screened for variants in CHEK2.CaseswithHBC mut software (Interactive Biosoftware, Rouen, France) linked to BRCA1 or BRCA2 mutations were excluded by [32]. Included were human (Homo sapiens)[GenBank: direct sequencing of both genes and by multiplex liga- NP_009125.1], chimpanzee (Pan troglodytes) [GenBank: tion-dependent probe amplification of BRCA1. A control XP_001172759.1], macaque (Macaca)[GenBank: group recruited from the same region of France con- XP_001101658.1], rat (Rattus norvegicus)[GenBank: sisted of 513 female volunteers in good health and with- NP_446129.1], mouse (Mus musculus)[GenBank: out any personal or family history of breast or NP_057890.1], dog (Canis lupus familiaris)[GenBank: gynecologic cancers at the time of the recruitment. All XP_543464.2], cow (Bos taurus)[GenBank: subjects signed informed consent agreements that were NP_001029703.1], chicken (Gallus gallus)[GenBank: approved by the CCPPRB Regional Ethics Committee XP_001232074.1], frog (Xenopus tropicalis)[GenBank: (Auvergne, France). To assess the relationship between NP_001119996.1] and pufferfish (Tetraodon nigroviridis) CHEK2 variants and breast cancer risk, logistic regres- [UniProtKB/TrEMBL:Q4TI84], all extracted from the sion was used to obtain odds ratios (as estimates of rela- Ensembl Compara database [35]. PolyPhen-2 score was tive risk) and 95% confidence intervals [28]. calculated online using default settings and accession numbers [UniProtKB/Swiss-Prot:O96017] [36,37]. The DNA extraction and sequencing potential impact on splicing was studied using SpliceSi- To identify variants in the CHEK2 gene, exons 2 to 14 teFinder, MaxEntScan and GeneSplicer prediction soft- were analyzed (exon 1 is noncoding, and exon 15, repre- ware [38-40]. senting 89 bp of coding sequence, could not be analyzed for all the patients, owing to the presence of repeated Plasmid constructs sequences) in both patients and controls for the geno- The pDream2.1 cloning vector (GenScript USA Inc, Pis- mic sequence [GenBank:NG_008150.1] and for the cataway, NJ, USA) carrying the full-length human cDNA sequence [GenBank:NM_007194.3] [29] DNA CHEK2 coding sequence tagged with an N-terminal was extracted from 10 ml of peripheral blood collected FLAG extension under the control of the LacZ Desrichard et al. Breast Cancer Research 2011, 13:R119 Page 3 of 11 http://breast-cancer-research.com/content/13/6/R119

promoter for expression in prokaryotes was verified to different variants in 16 of 507 cases and 4 different var- contain the wild-type (WT) sequence. The Stratagene iants in 4 of 513 controls (Table 1). In the case popula- QuickChange II Site-Directed Mutagenesis Kit (Agilent tion, there were eight different novel missense Technologies, Inc, Santa Clara, CA, USA) was used to mutations and one previously described in osteosarco- generate mutant constructs Ser39Phe, Pro85Arg, mas [41], as well as one nonsense mutation, one novel Arg117Gly, Arg145Trp, Glu161Del, Arg180His, Lys224- frame shift mutation, one splice donor mutation and Glu, Lys244Arg, Met367fsX15, Tyr390Ser and three patients (0.59%) with the c.1100delC Thr476Met, with the corresponding primers (available (Met367fsX13) mutation (Figure 1). No mutation hot- on request) in accordance with the manufacturer’s spots were observed (Figure 1). Mutations among con- recommendations. All constructs were confirmed by trols included three missense mutations and one sequencing of the entire coding region of the gene (pri- affecting a splice donor site. To the best of our knowl- mers available on request). edge, we are the first to report all mutations found in the control population. The missense mutation Expression and extraction of recombinant CHEK2 protein Lys244Arg was found in both cases and controls. The Escherichia coli strain BL21 was transformed with mutation frequency was higher for the cases (16 of pDream plasmids(GenScript USA Inc) encoding WT or 1,014 vs 4 of 1,026; P = 0.0065) (Table 2). The OR of mutated Flag-CHEK2. Cultures were grown at 37°C in CHEK2 mutation carriers was 4.15 (95% CI = 1.38 to Luria Broth media containing 100 μg/ml ampicillin until 12.50), suggesting that CHEK2 contributes to hereditary absorbance at 600 nm reached 0.6 before isopropyl b-D- risk of breast cancer. 1-thiogalactopyranoside was added to a final concentra- tion of 0.5 mM and incubated for 3 hours. Extraction of Bioinformatics study total bacterial proteins was performed as described pre- Canonical splice donor and acceptor sites were evalu- viously [26]. ated using SpliceSiteFinder, MaxEntScan and GeneSpli- cer. All three programs provided consistent information Kinase activity of CHEK2 recombinant proteins that the two mutations affecting splice donor sites abro- Omnia kinase assay buffer (18 μl; Invitrogen/Life Tech- gate splicing of the exons concerned (Table 1). We thus nologies, Carlsbad, CA, USA) containing 10 μM Sox sub- considered these mutations to be deleterious. Because strate peptide, 1 mM ATP, 0.2 mM dithiothreitol and 2 the effect of an amino substitution can be difficult to μl of 10 × Omnia buffer was incubated for 5 minutes at assess, a combination of three different in silico analyses room temperature and aliquoted to a 96-well plate to (Align-GVGD class, SIFT prediction and PolyPhen-2 ensure equal amounts of the chemosensor. For each prediction) was used. For each missense variation, we assay reaction, 1.5 μg of total bacterial protein from compiled these three scores to propose a diagnosis. Mis- induced cultures containing WT or mutated Flag- sense variants were considered probably deleterious if at CHEK2 or from untransformed E. coli, were then added least one deleterious score was obtained and probably and mixed gently. CHEK2 protein was added at the benign if three benign scores were obtained. Class above moment of the fluorescence acquisition, allowing us to C35 was considered the threshold for deleterious var- follow the kinetics of substrate phosphorylation. CHEK2 iants in Align-GVGD. kinase activity was monitored with excitation at 360 nm Substitutions with a SIFT score less than 0.05 are pre- and emission at 485 nm. Fluorescence was detected using dicted to be deleterious. A SIFT median sequence con- an Infinite 200 PRO plate reader (Tecan Group Ltd, servation score cutoff of 3.25 was used to measure the Männedorf, Switzerland) for 60 minutes at room tem- diversity of the sequences used for prediction, and a perature. For each mutation,anaverageofsixwellsand score greater than 3.25 could indicate that the predic- three independent experiments were conducted. Each tion was based on closely related sequences. This would curve was normalized by linear regression using the slope result in a low confidence score if the variant were con- of the corresponding nontransformed bacterial protein sidered deleterious. No SIFT median sequence conserva- extract curve. Thus the slope of the resulting curves tion score reached this cutoff, indicating that the aligned represents the ability of CHEK2 recombinant protein to sequences were diverse enough for confident prediction phosphorylate the substrate (see Additional file 1). of substitutions that should affect protein function. One mutation, Lys244Arg, present in both cases and con- Results trols, was not considered to be potentially deleterious CHEK2 mutations contribute to hereditary breast cancer on the basis of the results of any of the algorithms used, To evaluate the contribution of CHEK2 mutations to suggesting it is a rare but benign variant. All other mis- HBC, we sequenced the coding sequence of the gene, sense variants were considered potentially damaging on including intron-exon boundaries. We observed 13 the basis of at least one measure. Desrichard et al. Breast Cancer Research 2011, 13:R119 Page 4 of 11 http://breast-cancer-research.com/content/13/6/R119

Table 1 CHEK2 mutations identified in French women with hereditary breast cancer and a control group of unaffected women Mutation Amino acid Exon Protein Splicing Align- SIFT median sequence SIFT PolyPhen-2 Proposed change domain effect GVGD conservation score prediction prediction diagnosis class Case population (n = 13) 190G > A [49] Glu64Lys 2 SQ/TQ - C15 2.23 Tolerated Benign Probably (two cases) benign 190G > T Glu64X 2 SQ/TQ - - - - Probably deleterious 254C > G Pro85Arg 2 - - C0 2.15 Tolerated Probably Probably [23,41] damaging deleterious 349A > G [23] Arg117Gly 3 FHA - C65 2.07 Deleterious Probably Probably damaging deleterious 670A > G Lys224Glu 5 Kinase - C0 2.05 Tolerated Benign Probably benign 731A > G Lys244Arg 6 Kinase - C0 2.04 Tolerated Benign Probably benign 751A > T [23] Ile251Phe 6 Kinase - C0 2.04 Tolerated Probably Probably damaging deleterious 846+4_846 - 7 - Possibly - - - - Probably +7del skip exon 7 deleterious 1100del (three Thr367MetfsX15 11 Kinase - - - - - Probably cases) deleterious 1169A > C Tyr390Ser 11 Kinase - C0 2.04 Deleterious Probably Probably damaging deleterious 1281C > A Phe427Leu 12 Kinase - C0 2.04 Deleterious Probably Probably damaging deleterious 1427C > T [23] Thr476Met 13 Kinase - C0 2.05 Deleterious Probably Probably damaging deleterious Control population (n =4) 116C > T Ser39Phe 2 SQ/TQ - C65 3.10 Deleterious Benign Probably deleterious 251A > G Glu84Gly 2 - - C65 2.17 Tolerated Probably Probably damaging deleterious 731A > G Lys244Arg 6 Kinase - C0 2.04 Tolerated Benign Probably benign 792+1dup - 6 - Probably - - - - Probably skip exon 6 deleterious Additional mutations tested for kinase function [27] 410G > A Arg137Gln 3 FHA - C0 2.22 Tolerated Benign Probably benign 433C > T Arg145Trp 3 FHA - C65 2.05 Tolerated Probably Probably damaging deleterious 481_483del Glu161del 4 FHA - - - - - Probably deleterious 539G > A Arg180His 4 FHA - C0 2.04 Tolerated Probably Probably damaging deleterious Align-GVGD, SIFT (Sorting Intolerant From Tolerant) and PolyPhen-2 (Polymorphism Phenotyping v2) software tool scores and splicing effect were calculated. The possible impact of the different mutations on CHEK2 function is proposed. FHA = Forkhead-associated domain.

Effect of CHEK2 mutations on kinase activity Overexpression of recombinant CHEK2 at high levels in To evaluate whether missense variants inhibit the func- bacteria is associated with CHEK2 autophosphorylation tion of the CHEK2 protein, an in vitro kinase activity and activation in the absence of DNA damage [13]. This test based on a CHEK2-specific substrate peptide carry- property was used to obtain recombinant activated ing a C-terminal SOX was developed [42]. CHEK2. Upon the phosphorylation of the SOX-specific Desrichard et al. Breast Cancer Research 2011, 13:R119 Page 5 of 11 http://breast-cancer-research.com/content/13/6/R119

Figure 1 Position of CHEK2 mutations found in French non-BRCA HBC and control populations. FHA = Forkhead-associated domain; SQ/ TQ = SQ/TQ cluster domain. substrate by CHEK2, the presence of the chemosensor recombinant CHEK2 protein carrying c.1100delC (Fig- SOX results in an increase in fluorescence at 485 nm. ure2).Onlymissensevariantsweretestedforkinase Activity was detected for the WT protein but not for activity. c.190G > T (Glu64X), c.825_826del, c.846+4_ proteins extracted from nontransformed bacteria or +7del and c.792+1dup were considered deleterious

Table 2 CHEK2 mutations were more frequent in cases than in controls CHEK2 mutation type Controls Allelic frequency in Cases Allelic frequency in cases P value OR 95% CI (n = 1,026)a controls (n = 1,014)a Any CHEK2 mutation 4 0.39% 16 1.58% 0.0065 4.15 (1.38 to 12.50) CHEK2 deleterious 3 0.29% 15 1.48% 0.0042 5.18 (1.49 to 18.00) mutations an indicates the number of alleles. Desrichard et al. Breast Cancer Research 2011, 13:R119 Page 6 of 11 http://breast-cancer-research.com/content/13/6/R119

Figure 2 Kinase activity of recombinant Flag-CHEK2 protein.Totalproteinextract(1.5μg) was tested for the ability to phosphorylate a fluorescent substrate. The slope of the resulting curve represents Flag-CHEK2 kinase activity. The slope of the wild-type (WT) Flag-CHEK2 kinase activity curve was normalized to 1. Nontransformed protein extracts (NT) and mutant c.1100delC served as controls. Each point represents an average of five measurements performed in triplicate. without further analysis. Four mutations tested for 2). The mutants Pro85Arg, Arg180His and Lys244Arg kinase activity in vitro by Sodha et al. [27] were had significantly lower, but not null, kinase activity (Fig- included to validate the assay. ure 2), which placed them in the intermediate class. The Three different classes of kinase activity were mutations Glu161Del, Lys224Glu, Thr476Met and observed: WT-like, intermediate and null (Figure 2). Tyr380Ser did not have any kinase activity. Nine of the Mutations Ser39Phe, Arg145Trp and Arg137Gln exhib- eleven mutations showed kinase activity consistent with ited WT-like kinase activity, suggesting that these muta- the in silico analysis, demonstrating the good but tions do not affect the ability of recombinant CHEK2 to incomplete correlation of those two approaches (Table recognize, bind and phosphorylate its substrate (Figure 3).

Table 3 Relationship between in silico and in vitro results Mutation Domain In silico conclusion In vitro conclusion Concordance Ser39Phe SQ/TQ Probably deleterious WT-like No Pro85Arg Unknown function Probably deleterious Intermediate Yes Arg137Gln FHA Probably benign WT-like Yes Arg145Trp FHA Probably deleterious Intermediate Yes Glu161Del FHA Probably deleterious Null Yes Arg180His FHA Probably deleterious Intermediate Yes Lys 224Glu Kinase core Probably benign Null No Lys244Arg Kinase core Probably benign WT-like Yes Met367fsX13 Kinase core Probably deleterious Null Yes Tyr390Ser Kinase core Probably deleterious Null Yes Thr476Met Kinase core Probably deleterious Null Yes Desrichard et al. Breast Cancer Research 2011, 13:R119 Page 7 of 11 http://breast-cancer-research.com/content/13/6/R119

Both in silico and in vitro analyses suggested that the coding sequence was necessary to capture the majority variant Lys244Arg, present in cases and controls, can be of the different mutations present. This might be the considered benign. This variant was thus removed from case for other populations where the frequency of the the pool of potentially deleterious CHEK2 variants that CHEK2 founder mutations is low. contribute to HBC. As a result, the mutation frequency In Table 4, to give an overview of CHEK2 contribution was reduced to 1.48% for cases and 0.29% for controls to breast cancer, we summarize the results of 36 different (Table 2). This difference remained significant (P = case-control studies from different countries where the 0.0042), and the OR associated with the presence of a presence of variants was assessed by allele-specific deleterious mutation was increased to 5.18 (95% CI: sequencing or DNA sequencing of the entire gene. The 1.49 to 18.00). ORs of breast cancer from the different studies of c.1100delC are similar, regardless of the selection of Discussion cases, with a combined OR of 2.77. We also found com- We found strong evidence of an association between parable results for the other protein-truncating mutation CHEK2 variants and HBC, with an OR of 5.18. Of 16 c.444+1G > A, which is less frequent but has an OR simi- different mutations, 9 were unreferenced variants. This lar to that for c.1100delC. No positive association with demonstrates that, in populations without founder HBC was observed, possible due to the very low fre- mutations, an aggregate of rare variants makes CHEK2 quency of the variant in both cases and controls. The fre- an appreciable breast cancer risk gene. quent variant I157T was associated with lower ORs than The functional consequences of missense variants can null mutations. Although this variant has been associated be difficult to establish, and in estimating associated with breast cancer risk in early-onset or unselected cases, risks it is important to separate deleterious from neutral in our study it did not exhibit a significant association variants. We were unfortunately unable to complement with HBC. Although the frequency of these deleterious the functional data presented here with a study of the mutations was different among populations, the ORs cosegregation of these variants with cancer, because associated with breast cancer were consistent for the two only the index case was available for analysis in the null mutations and lower for the missense mutation. majority of families. These data were collected using allele-specific sequen- Missense variant Ser39Phe was predicted as probably cing, suggesting that testing for CHEK2 founder muta- deleterious by two of the three scores (SIFT and Align- tions is cost-effective in some populations because the GVGD), but exhibited WT-like kinase activity. This dis- variants are sufficiently common and the test is relatively cordance may suggest that not all deleterious changes in inexpensive. Consequently, however, these techniques the CHEK2 protein can be revealed by the in vitro exclude mutations present elsewhere in the gene. kinase activity test, most notably for changes outside the Because the c.1100delC allele does not seem to be catalytic domain. Changes affecting interactions with present in southern Europeans or in most non-Cauca- upstream activators such as ATM, for example, may not sian populations [45-47], other research groups have be detectable by our measure. In contrast, Lys224Glu used full-gene sequencing to determine whether other was predicted to be a tolerable change by the three variants contribute to breast cancer risk. There is a posi- scores, but exhibited null kinase activity, demonstrating tive association between CHEK2 variants and HBC in the complementarity of those two approaches. the Australian, Canadian, North American, German and Further functional tests, such as expression in eukar- now French, but not Czech Republic, populations yotic cells, followed by measures of activation by DNA [22-25]. This suggests that CHEK2 analysis in popula- strand breaks, protein stability and interaction with cel- tions where the common founder mutations are rare lular partners may be necessary to appreciate all effects requires screening of the entire sequence. of these mutations, especially for those where the in Narod’s [48] recent review supports the view that test- silico and in vitro conclusions differ. We thus retain this ing non-BRCA HBC families for mutations in CHEK2 variant as potentially deleterious, unlike Lys244Arg, can provide useful information to evaluate the risk of which was characterized as benign by all measures. breast cancer and suggests that the relatively high cost The association between the CHEK2 gene and breast of sequencing makes only the targeted search of fre- cancer risk has been supported mainly by case-control quent mutations cost-effective. In certain populations, studies of founder mutations such as 1100delC, I157T one or a few mutations do indeed capture the majority (frequent in northern and eastern Europe) or the Polish of CHEK2 variants associated with cancer risk. In most founder mutation IVS2+1 G > A (c.444+1G > A) regions, however, this allele-specific approach is inade- [19,20,24,43,44]. In our population, only one of these quate and a full-resequencing strategy should be consid- founder mutations was observed, accounting for one- ered. The rapidly falling cost of resequencing, as well as third of deleterious mutations. Analysis of the entire alternate techniques, should make this possible. Desrichard et al. Breast Cancer Research 2011, 13:R119 Page 8 of 11 http://breast-cancer-research.com/content/13/6/R119

Table 4 Odds ratio for breast cancer among women with CHEK2 variants Variants References Geographic populations Case Mutation frequency Mutation frequency OR (95% populations in controls (n/total) in cases (n/total) (CI) c.1100delC Mixed populations All studies 0.33% (559/166,596) 0.90% (861/94,076) 2.77 (2.49 to 3.08)** [19,22,25,45,47,50-59] AJ, AUS, B, CDN, CZ, DK, E, FIN, UBC 0.31% (2,502/80,168) 0.80% (464/58,290) 2.56 (2.19 G, IRL, KP, NL, S, UK, USA to 2.99)** [3,19,22,24,25,50,54,60-64] B, CDN, CZ, D, FIN, NL, S, UK, HBC 0.39% (111/28,402) 1.27% (215/17,000) 3.29 (2.61 USA to 4.14)** [23,54,65] D, USA, AUS, CDN EOBC 0.14% (14/9,846) 0.46% (21/4,588) 2.77 (1.23 to 6.26)** [18,53,60,66,67] AJ, DK, G, FIN, NL, RUS, UK, USA BBC 0.43% (130/29,936) 1.35% (142/10,496) 3.17 (2.49 to 4.03)** c.444+1G > Mixed populations All studies 0.19% (82/42,266) 0.66% (217/33,142) 3.45 (2.67 A to 4.46)** (IVS2+1G > A) [68,69] PL EOBC 0.19% (49/25,426) 0.59% (91/15,338) 3.10 (2.19 to 4.39)** [24,43] D, BY HBC 0.12% (3/2,586) 0.26% (4/1,536) 2.25 (0.5 to 10.08) (NS) [43,59,70] D, BY, PL UBC 0.20% (28/14,254) 0.61% (113/18,604) 3.12 (2.06 to 4.72)** I157T Mixed populations All studies 2.14% (1,031/48,268) 3.11% (1062/34,128) 1.56 (1.43 to 1.70)** [22,23,65,68] PL, USA, AUS, CDN EOBC 1.46% (270/18,432) 1.99% (215/10,780) 1.59 (1.32 to 1.91)** [20,22,24,43,71] D, BY, FIN, NL, USA HBC 1.77% (116/6,544) 1.51% (47/3,118) 0.89 (0.60 to 1.20) (NS) [20,22,25,43,44,59,70] By, CZ, D, FIN, PL, USA UBC 2.04% (644/31,600) 2.95% (807/27,346) 1.48 (1.33 to 1.65)** Whole- [22] USA UBC 0.57% (24/4,210) 0.50% (4/800) 0.88 (0.3 to gene 2.55) (NS) studies [24] D HBC 0.50% (18/3,630) 3.00% (31/1,032) 6.38 (3.54 to 11.5)** [25] CZ HBC 1.39% (19/1,366) 2.01% (27/1,346) 1.46 (0.80 to 2.65) (NS) F HBC 0.39% (4/1,026) 1.58% (16/1,014) 4.15 (1.38 to 12.50)** [23] USA, CDN, AUS EOBC 1.84% (40/2,178) 4.91% (6/2,484) 2.76 (1.65 to 4.60)** BBC = bilateral breast cancer; EOBC = early-onset breast cancer; HBC = hereditary breast cancer not related to a BRCA mutation; UBC = unselected breast cancer; AJ = Ashkenazi Jewish; AUS = Australia; B = Belgium; BY = Belarus; CDN = Canada; CZ = Czech Republic; D = Germany; DK = Denmark; E = Spain; F = France; FIN = Finland; IRL = Ireland; KP = South Korea; NL = The Netherlands; PL = Poland; RUS = Russia; S = Sweden; UK = United Kingdom; USA = United States of America; NS = nonsignificant (P < 0.05); **P < 0.0001. Participant numbers may vary from original publications because of exclusion of study subgroups.

Conclusions understanding of the contribution of this gene to heredi- The usefulness of the information gained from genetic tary cancer risk. analysis of CHEK2 is currently a matter of debate. As we have discussed, the risk of breast cancer for a Web resources woman with a null mutation in this gene is increased The URLs for the accession numbers and data presented two- to fivefold. Increased breast surveillance may be herein are as follows: proposed for carriers, but when counseling a family with Entrez gene database: http://www.ncbi.nlm.nih.gov/ many breast cancer cases, only some of whom carry the gene (for CHEK2 sequencing) CHEK2 mutation, it is unclear what advice may be given Ensembl Compara database: http://www.ensembl.org/ to noncarriers. Collecting research information on info/docs/compara/index.html CHEK2 mutations, however, serves to advance our Desrichard et al. Breast Cancer Research 2011, 13:R119 Page 9 of 11 http://breast-cancer-research.com/content/13/6/R119

UniProtKB/Swiss-Prot database: http://www.uniprot. ATM mutations that cause ataxia-telangiectasia are breast cancer org/uniprot susceptibility alleles. Nat Genet 2006, 38:873-875. 5. Seal S, Thompson D, Renwick A, Elliott A, Kelly P, Barfoot R, Chagtai T, Jayatilake H, Ahmed M, Spanova K, North B, McGuffog L, Evans DG, Additional material Eccles D, Breast Cancer Susceptibility Collaboration (UK), Easton DF, Stratton MR, Rahman N: Truncating mutations in the Fanconi anemia J gene BRIP1 are low-penetrance breast cancer susceptibility alleles. Nat Additional file 1: Supplementary data: control kinase activity of Genet 2006, 38:1239-1241. recombinant CHEK2 protein. Total protein extract (1.5 μg) was added 6. Antoniou AC, Pharoah PD, McMullan G, Day NE, Ponder BA, Easton D: to the substrate peptide. Fluorescence was measured at 485 nm for 1 Evidence for further breast cancer susceptibility genes in addition to hour. Wild type (WT), nontransformed protein extracts (NT) and mutant BRCA1 and BRCA2 in a population-based study. Genet Epidemiol 2001, c.1100delC served as controls, and kinase activity of bacterially expressed 21:1-18. mutants are sorted by domain. Each point on the curve represents an 7. Ponder BA: Cancer genetics. Nature 2001, 411:336-341. average of six measurements repeated in triplicate. 8. Antoniou AC, Pharoah PD, McMullan G, Day NE, Stratton MR, Peto J, Ponder BJ, Easton DF: A comprehensive model for familial breast cancer incorporating BRCA1, BRCA2 and other genes. Br J Cancer 2002, 86:76-83. 9. Pharoah PD, Antoniou A, Bobrow M, Zimmern RL, Easton DF, Ponder BA: Abbreviations Polygenic susceptibility to breast cancer and implications for prevention. HBC: hereditary breast cancer; FHA: Forkhead-associated; WT: wild type. Nat Genet 2002, 31:33-36. 10. Sodha N, Houlston RS, Bullock S, Yuille MA, Chu C, Turner G, Eeles RA: CHEK2 Acknowledgements Increasing evidence that germline mutations in do not cause Li- We thank Molecular Genetic Diagnosis team and Dr Bernard-Gallon’s team Fraumeni syndrome. Hum Mutat 2002, 20:460-462. for technical assistance. The Ligue Contre le Cancer d’Auvergne provided 11. Bartek J, Falck J, Lukas J: Chk2 kinase: a busy messenger. Nat Rev Mol Cell financial support. A. Desrichard was funded by grants from the Conseil Biol 2001, 2:877-886. Regional d’Auvergne and FEDER. 12. Matsuoka S, Huang M, Elledge SJ: Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science 1998, 282:1893-1897. Author details 13. Ahn JY, Li X, Davis HL, Canman CE: Phosphorylation of threonine 68 1Laboratoire Diagnostic Génétique et Moléculaire, Centre Jean Perrin, 58 rue promotes oligomerization and autophosphorylation of the Chk2 protein Montalembert, F-63011 Clermont-Ferrand, France. 2Clermont Université, kinase via the Forkhead-associated domain. J Biol Chem 2002, Université d’Auvergne, 28 place Henri Dunant, EA 4233, BP 10448, F-63001 277:19389-19395. Clermont Ferrand, France. 14. Lee JS, Collins KM, Brown AL, Lee CH, Chung JH: hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response. Nature Authors’ contributions 2000, 404:201-204. AD contributed to the sequencing of CHEK2, designed and performed the 15. Falck J, Mailand N, Syljuåsen RG, Bartek J, Lukas J: The ATM-Chk2-Cdc25A kinase activity, participated in the in silico analyses and drafted the checkpoint pathway guards against radioresistant DNA synthesis. Nature manuscript. 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doi:10.1186/bcr3062 Cite this article as: Desrichard et al.: CHEK2 contribution to hereditary breast cancer in non-BRCA families. Breast Cancer Research 2011 13:R119.

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